Wnt Planar Cell Polarity Research Articles

Making a Tubule the Noncanonical Way

Tubulogenesis is an essential feature during formation of multiple organ systems. The genitourinary, cardiovascular, auditory, pulmonary, and gastrointestinal systems all require the formation of epithelialized tubes for proper function. Interest in the developmental mechanisms controlling this

Getting to the heart of planar cell polarity signaling

The genes that underpin normal heart development, and which can be disrupted to result in congenital structural malformations, are rapidly being uncovered. However, the specific cellular processes that lie downstream of these genetic cascades, accurately shaping tissues and complex structures within the heart, remain relatively unclear. The noncanonical Wnt planar cell polarity (PCP) signaling pathway is known to have a role in embryonic morphogenesis and as such is an important candidate pathway to carry out these roles in heart development. The pathway regulates the polarization of cells in a variety of contexts, allowing cells to change shape and position and to "know" their orientation within a mass of tissue. PCP signaling has also been shown recently to regulate the cellular position of the primary cilium. This organelle is known to be crucial for the establishment of left-right patterning in the early embryo and may also act as a signaling antenna for other developmental and regulatory pathways. It is not surprising that recent studies have also linked PCP to left-right patterning. In this review, we will examine the current evidence suggesting that PCP signaling has a central role in cardiac development and malformation.

Control of the Wnt pathways by nephrocystin-4 is required for morphogenesis of the zebrafish pronephros

Nephronophthisis is a hereditary nephropathy characterized by interstitial fibrosis and cyst formation. It is caused by mutations in NPHP genes encoding the ciliary proteins, nephrocystins. In this paper, we investigate the function of nephrocystin-4, the product of the nphp4 gene, in vivo by morpholino-mediated knockdown in zebrafish and in vitro in mammalian kidney cells. Depletion of nephrocystin-4 results in convergence and extension defects, impaired laterality, retinal anomalies and pronephric cysts associated with alterations in early cloacal morphogenesis. These defects are accompanied by abnormal ciliogenesis in the cloaca and in the laterality organ. We show that nephrocystin-4 is required for the elongation of the caudal pronephric primordium and for the regulation of cell rearrangements during cloaca morphogenesis. Moreover, depletion of either inversin, the product of the nphp2 gene, or of the Wnt-planar cell polarity (PCP) pathway component prickle2 increases the proportion of cyst formation in nphp4-depleted embryos. Nephrocystin-4 represses the Wnt-β-catenin pathway in the zebrafish cloaca and in mammalian kidney cells in culture. In these cells, nephrocystin-4 interacts with inversin and dishevelled, and regulates dishevelled stability and subcellular localization. Our data point to a function of nephrocystin-4 in a tight regulation of the Wnt-β-catenin and Wnt-PCP pathways, in particular during morphogenesis of the zebrafish pronephros. Moreover, they highlight common signalling functions for inversin and nephrocystin-4, suggesting that these two nephrocystins are involved in common physiopathological mechanisms.

Wnt5a Regulates Midbrain Dopaminergic Axon Growth and Guidance

During development, precise temporal and spatial gradients are responsible for guiding axons to their appropriate targets. Within the developing ventral midbrain (VM) the cues that guide dopaminergic (DA) axons to their forebrain targets remain to be fully elucidated. Wnts are morphogens that have been identified as axon guidance molecules. Several Wnts are expressed in the VM where they regulate the birth of DA neurons. Here, we describe that a precise temporo-spatial expression of Wnt5a accompanies the development of nigrostriatal projections by VM DA neurons. In mice at E11.5, Wnt5a is expressed in the VM where it was found to promote DA neurite and axonal growth in VM primary cultures. By E14.5, when DA axons are approaching their striatal target, Wnt5a causes DA neurite retraction in primary cultures. Co-culture of VM explants with Wnt5a-overexpressing cell aggregates revealed that Wnt5a is capable of repelling DA neurites. Antagonism experiments revealed that the effects of Wnt5a are mediated by the Frizzled receptors and by the small GTPase, Rac1 (a component of the non-canonical Wnt planar cell polarity pathway). Moreover, the effects were specific as they could be blocked by Wnt5a antibody, sFRPs and RYK-Fc. The importance of Wnt5a in DA axon morphogenesis was further verified in Wnt5a −/− mice, where fasciculation of the medial forebrain bundle (MFB) as well as the density of DA neurites in the MFB and striatal terminals were disrupted. Thus, our results identify a novel role of Wnt5a in DA axon growth and guidance.

BMP promotes motility and represses growth of smooth muscle cells by activation of tandem Wnt pathways

We present a novel cell-signaling paradigm in which bone morphogenetic protein 2 (BMP-2) consecutively and interdependently activates the wingless (Wnt)-β-catenin (βC) and Wnt-planar cell polarity (PCP) signaling pathways to facilitate vascular smooth muscle motility while simultaneously suppressing growth. We show that BMP-2, in a phospho-Akt-dependent manner, induces βC transcriptional activity to produce fibronectin, which then activates integrin-linked kinase 1 (ILK-1) via α4-integrins. ILK-1 then induces the Wnt-PCP pathway by binding a proline-rich motif in disheveled (Dvl) and consequently activating RhoA-Rac1-mediated motility. Transfection of a Dvl mutant that binds βC without activating RhoA-Rac1 not only prevents BMP-2-mediated vascular smooth muscle cell motility but promotes proliferation in association with persistent βC activity. Interfering with the Dvl-dependent Wnt-PCP activation in a murine stented aortic graft injury model promotes extensive neointima formation, as shown by optical coherence tomography and histopathology. We speculate that, in response to injury, factors that subvert BMP-2-mediated tandem activation of Wnt-βC and Wnt-PCP pathways contribute to obliterative vascular disease in both the systemic and pulmonary circulations.

Wnt/PCP signaling controls intracellular position of MTOCs during gastrulation convergence and extension movements

During vertebrate gastrulation, convergence and extension cell movements are coordinated with the anteroposterior and mediolateral embryonic axes. Wnt planar cell polarity (Wnt/PCP) signaling polarizes the motile behaviors of cells with respect to the anteroposterior embryonic axis. Understanding how Wnt/PCP signaling mediates convergence and extension (C&E) movements requires analysis of the mechanisms employed to alter cell morphology and behavior with respect to embryonic polarity. Here, we examine the interactions between the microtubule cytoskeleton and Wnt/PCP signaling during zebrafish gastrulation. First, we assessed the location of the centrosome/microtubule organizing center (MTOC) relative to the cell nucleus and the body axes, as a marker of cell polarity. The intracellular position of MTOCs was polarized, perpendicular to the plane of the germ layers, independently of Wnt/PCP signaling. In addition, this position became biased posteriorly and medially within the plane of the germ layers at the transition from mid- to late gastrulation and from slow to fast C&E movements. This depends on intact Wnt/PCP signaling through Knypek (Glypican4/6) and Dishevelled components. Second, we tested whether microtubules are required for planar cell polarization. Once the planar cell polarity is established, microtubules are not required for accumulation of Prickle at the anterior cell edge. However, microtubules are needed for cell-cell contacts and initiation of its anterior localization. Reciprocal interactions occur between Wnt/PCP signaling and microtubule cytoskeleton during C&E gastrulation movements. Wnt/PCP signaling influences the polarity of the microtubule cytoskeleton and, conversely, microtubules are required for the asymmetric distribution of Wnt/PCP pathway components.

Structure and expression of conserved Wnt pathway components in the demosponge Amphimedon queenslandica

Wnt-signalling plays a critical role in animal development, and its misregulation results in serious human diseases, including cancer. While the Wnt pathway is well studied in eumetazoan models, little is known about the evolutionary origin of its components and their functions. Here, we have identified key machinery of the Wnt-β-catenin (canonical)-signalling pathway that is encoded in the Amphimedon queenslandica (Demospongiae; Porifera) genome, namely Wnt, Fzd, SFRP, Lrp5/6, Dvl, Axin, APC, GSK3, β-catenin, Tcf, and Groucho. Most of these genes are not detected in the choanoflagellate and other nonmetazoan eukaryotic genomes. In contrast, orthologues of some of key components of bilaterian Wnt-planar cell polarity and Wnt/Ca(2+) are absent from the Amphimedon genome, suggesting these pathways evolved after demosponge and eumetazoan lineages diverged. Sequence analysis of the identified proteins of the Wnt-β-catenin pathway has revealed the presence of most of the conserved motifs and domains responsible for protein-protein and protein-DNA interactions in vertebrates and insects. However, several protein-protein interaction domains appear to be absent from the Amphimedon Axin and APC proteins. These are also missing from their orthologues in the cnidarian Nematostella vectensis, suggesting that they are bilaterian novelties. All of the analyzed Wnt pathway genes are expressed in specific patterns during Amphimedon embryogenesis. Most are expressed in especially striking and highly dynamic patterns during formation of a simple organ-like larval structure, the pigment ring. Overall, our results indicate that the Wnt-β-catenin pathway was used in embryonic patterning in the last common ancestor of living metazoans. Subsequently, gene duplications and a possible increase in complexity of protein interactions have resulted in the precisely regulated Wnt pathway observed in extant bilaterian animals.

Vang-like protein 2 and Rac1 interact to regulate adherens junctions

The Wnt planar cell polarity (Wnt/PCP) pathway signals through small Rho-like GTPases to regulate the cytoskeleton. The core PCP proteins have been mapped to the Wnt/PCP pathway genetically, but the molecular mechanism of their action remains unknown. Here, we investigate the function of the mammalian PCP protein Vang-like protein 2 (Vangl2). RNAi knockdown of Vangl2 impaired cell-cell adhesion and cytoskeletal integrity in the epithelial cell lines HEK293T and MDCK. Similar effects were observed when Vangl2 was overexpressed in HEK293T, MDCK or C17.2 cells. The effects of Vangl2 overexpression could be blocked by knockdown of the small GTPase Rac1 or by dominant-negative Rac1. In itself, knockdown of Rac1 impaired cytoskeletal integrity and reduced cell-cell adhesion. We found that Vangl2 bound and re-distributed Rac1 within the cells but did not alter Rac1 activity. Moreover, both transgenic mouse embryos overexpressing Vangl2 in neural stem cells and loop-tail Vangl2 loss-of-function embryos displayed impaired adherens junctions, a cytoskeletal unit essential for neural tube rigidity and neural tube closure. In vivo, Rac1 was re-distributed within the cells in a similar way to that observed by us in vitro. We propose that Vangl2 affects cell adhesion and the cytoskeleton by recruiting Rac1 and targeting its activity in the cell to adherens junctions.

Vang-like protein 2 and Rac1 interact to regulate adherens junctions

The Wnt planar cell polarity (Wnt/PCP) pathway signals through small Rho-like GTPases to regulate the cytoskeleton. The core PCP proteins have been mapped to the Wnt/PCP pathway genetically, but the molecular mechanism of their action remains unknown. Here, we investigate the function of the mammalian PCP protein Vang-like protein 2 (Vangl2). RNAi knockdown of Vangl2 impaired cell-cell adhesion and cytoskeletal integrity in the epithelial cell lines HEK293T and MDCK. Similar effects were observed when Vangl2 was overexpressed in HEK293T, MDCK or C17.2 cells. The effects of Vangl2 overexpression could be blocked by knockdown of the small GTPase Rac1 or by dominant-negative Rac1. In itself, knockdown of Rac1 impaired cytoskeletal integrity and reduced cell-cell adhesion. We found that Vangl2 bound and re-distributed Rac1 within the cells but did not alter Rac1 activity. Moreover, both transgenic mouse embryos overexpressing Vangl2 in neural stem cells and loop-tail Vangl2 loss-of-function embryos displayed impaired adherens junctions, a cytoskeletal unit essential for neural tube rigidity and neural tube closure. In vivo, Rac1 was re-distributed within the cells in a similar way to that observed by us in vitro. We propose that Vangl2 affects cell adhesion and the cytoskeleton by recruiting Rac1 and targeting its activity in the cell to adherens junctions.

Wnt Pathway Regulation in Chronic Renal Allograft Damage

The Wnt signaling pathway, linked to development, has been proposed to be recapitulated during the progressive damage associated with chronic organ failure. Chronic allograft damage following kidney transplantation is characterized by progressive fibrosis and a smoldering inflammatory infiltrate. A modified, Fischer 344 (RT1(lvl)) to Lewis (RT1(l)) rat renal allograft model that reiterates many of the major pathophysiologic processes seen in patients with chronic allograft failure was used to study the progressive disease phenotype and specific gene product expression by immunohistochemistry and transcriptomic profiling. Central components of the Tgfb, canonical Wnt and Wnt-Ca2+ signaling pathways were significantly altered with the development of chronic damage. In the canonical Wnt pathway, Wnt3, Lef1 and Tcf1 showed differential regulation. Target genes Fn1, Cd44, Mmp7 and Nos2 were upregulated and associated with the progression of renal damage. Changes in the Wnt-Ca2+ pathway were evidenced by increased expression of Wnt6, Wnt7a, protein kinase C, Cam Kinase II and Nfat transcription factors and the target gene vimentin. No evidence for alterations in the Wnt planar cell polarity (PCP) pathway was detected. Overall results suggest cross talk between the Wnt and Tgfb signaling pathways during allograft inflammatory damage and present potential targets for therapeutic intervention.

The planar cell polarity pathway directs parietal endoderm migration

Parietal endoderm (PE) contributes to the yolk sac and is the first migratory cell type in the mammalian embryo. We can visualize PE migration in vitro using the F9 teratocarcinoma derived embryoid body outgrowth system and, show here that PE migration is directed by the non-canonical Wnt planar cell polarity (PCP) pathway via Rho/ROCK. Based on golgi apparatus localization and microtubule orientation, 68.6% of cells in control outgrowths are oriented in the direction of migration. Perturbation of Wnt signaling via sFRP treatment results in a loss of orientation coupled with an increase in cell migration. Inhibition of the PCP pathway at the level of Daam1 also results in a loss of cell orientation along with an increase in cell migration, as seen with sFRP treatment. Constitutively active Daam can inhibit the loss of orientation that occurs with sFRP treatment. We previously demonstrated that ROCK inhibition leads to an increase in cell migration, and we now show that these cells also lack oriented migration. Canonical Wnt signaling or the Rac arm of the PCP pathway does not appear to play a role in PE oriented migration. These data suggest the PCP pathway via Rho/ROCK modulates migration of PE.

Sfrp1, Sfrp2, and Sfrp5 regulate the Wnt/β‐catenin and the planar cell polarity pathways during early trunk formation in mouse

AbstractThe secreted frizzled‐related protein gene family encodes proteins that regulate Wnt signaling. Msx1 in situ hybridization of 9.5 days post coitus mouse embryos showing normal neural tube development in an Sfrp1; Sfrp2 double mutant (left) but severe neural tube defects in a Looptail (Lp/+); Sfrp1; Sfrp2 triple mutant (right). These findings suggest that Sfrps regulate the Wnt planar cell polarity pathway. See Satoh et al. in this issue.

Brachyury‐downstream gene sets in a chordate, Ciona intestinalis: integrating notochord specification, morphogenesis and chordate evolution

In vertebrates, Brachyury, a T-box transcription factor gene, seems to have a dual role in the differentiation of axial midline mesoderm cells into notochord and gastrulation cell movements regulated by non-canonical Wnt/planar cell polarity (Wnt/PCP) signaling. To understand the function of Brachyury-downstream genes in chordate embryos, from a series of our survey on differential expression, including subtractive hybridization, dot-blot assays, EST sequences and the expression patterns in whole-mount in situ hybridization at embryonic stages, we developed a knowledge database called "CINOBI: CionaNotochord and Brachyury-downstream gene Index" to create comprehensive catalogues of Brachyury-downstream gene sets in Ciona intestinalis. Combining genome and large-scale cDNA data, we were able to characterize 450 non-redundant Brachyury-downstream genes: Twenty-four genes were newly annotated as notochord-expressed genes. Several genes are components of signaling pathways such as Wnt/PCP, Nf kappaB and TGF-beta signaling. We propose that Brachyury is linked to these pathways regulating the expression of each component, and such a regulatory mechanism might be conserved among chordates.

C-Jun N-terminal kinase (JNK) cooperates with Gsk3β to regulate Dishevelled-mediated microtubule stability

BackgroundWnt factors are a large family of signaling molecules that play important roles in the regulation of cell fate specification, tissue polarity and cell movement. In the nervous system, Wnts also regulates the formation of neuronal connection acting as retrograde signals that regulate the remodeling of the axons prior to the assembly of the presynaptic apparatus. The scaffold protein Dishevelled (Dvl) mimics the effect of Wnt on the neuronal cytoskeleton by increasing the number of stable microtubule along the axon shaft and inducing the formation of looped microtubules (MT) at enlarged growth cones. A divergent Wnt-Dvl canonical pathway which bifurcates downstream of Gsk3β regulates MT dynamics.ResultsHere we show that the Wnt pathway also activates c-Jun N-terminal kinase (JNK) to regulate MT stabilization. Although in the Wnt planar cell polarity (PCP) pathway, JNK lays downstream of Rho GTPases, these GTPases are not required for Wnt-mediated MTs stability. Epistatic analyses and pharmacological studies suggest that the Wnt-Dvl signalling regulates the dynamic of the cytoskeleton through two different pathways that lead to inhibition of Gsk3β and activation of JNK in the same cell.ConclusionWe demonstrate a novel role for JNK in Wnt-mediated MT stability. Wnt-Dvl pathway increases MT stability through a transcription independent mechanism that requires the concomitant inhibition of Gsk3β and activation of JNK. These studies demonstrate that Wnts can simultaneously activate different signalling pathways to modulate cytoskeleton dynamics.

The Wnt inhibitor, Dickkopf 4, is induced by canonical Wnt signaling during ectodermal appendage morphogenesis

Ectodermal appendage morphogenesis requires continuous epithelial–mesenchymal cross-talk during development. Canonical Wnt signaling has been shown to be pivotal during this process and its inhibition leads to the absence of any morphological or molecular signs of appendage formation, including hair follicles (HFs). In the mouse, primary HFs arise in utero starting just before E14.5, when the first morphological signs of a placode are discernible. In this study, our goal was to identify novel factors expressed during primary HF morphogenesis. We performed transcriptional profiling of the developing epidermis at 12 h intervals between E12.5 and E15.5. One of the significantly differentially expressed genes was the Wnt inhibitor Dickkopf 4, Dkk4. We show that Dkk4 mRNA increases sharply in the dorso-lateral epidermis around E14 and then decreases until E15.5. Using whole mount in situ hybridization, we show that Dkk4 mRNA is localized to the pre-placodes at sites of presumptive epithelial–mesenchymal interactions during appendage morphogenesis, including the dental lamina, mammary gland, eccrine gland, and primary and secondary HFs. In silico analysis, reporter gene assays as well as in vitro transfections of LEF1 and β-catenin show that Dkk4 is a potential downstream target of canonical Wnt signaling. In addition, we demonstrate a direct physical interaction between LEF1/β-catenin complex and the Dkk4 promoter using ChIP. We propose that Dkk4 acts in a negative feedback loop to attenuate canonical Wnt signaling, and may facilitate a switch to the non-canonical Wnt planar cell polarity (PCP) pathway that is involved in cell movements during morphogenesis.

Wnt, a Driver of Myocardialization?

See related article, pages 292–299 In this current issue of Circulation Research , Phillips and coworkers1 carefully analyze the cardiovascular defect observed in the naturally occurring Loop-tail ( Lp ) mouse strain, which is best known for its severe neural tube defects.2 The genetic alteration in the Lp mouse was identified several years ago by two independent groups and appears to be a missense mutation in the Van Gogh–like 2 ( VANGL2 ) gene (S464N).3,4 Moreover, a second mouse strain was identified with another mutation in the VANGL2 gene (D255E) which has a similar phenotype.5 Vangl2 encodes a 521-aa protein which is predicted to contain four transmembrane regions, both the N and C termini protruding into the cytoplasm, and a C-terminal PDZ domain. The ancestral gene, Van Gogh ( VANG ), was initially identified in Drosophila as a regulator of cell/tissue polarity during development. Cell polarization is coordinated not only by Vang/Vangl but also by other components of the Wnt-Frizzled signaling pathway (see reviews6,7). Which Wnt growth factors are involved in triggering the signal transduction is not yet known, though Wnt11 is a potential candidate based on zebrafish studies.8 In vertebrates a second homologue of Vang was cloned, which is referred to as Van Gogh–like 1. Vangl1 and 2 are structurally similar. In vitro assays showed that both Vangl1 and 2 are able to directly interact through their PDZ domain with all three known members of the Wnt intracellular signal transducer Deshevelled (Dvl). Moreover, both known Vangl2 mutations were found to disrupt binding to Dvl, with Dvl1 being most sensitive.9 Dvl is not specifically involved in the Wnt-planar cell polarity (PCP) pathway, but was also found to transduce signals in the canonical Wnt-β-catenin and the non-canonical Wnt-Ca2+ pathway. The group of Dr Henderson had previously shown that …

STAT3 noncell-autonomously controls planar cell polarity during zebrafish convergence and extension.

Zebrafish signal transducer and activator of transcription 3 (STAT3) controls the cell movements during gastrulation. Here, we show that noncell-autonomous activity of STAT3 signaling in gastrula organizer cells controls the polarity of neighboring cells through Dishevelled-RhoA signaling in the Wnt-planar cell polarity (Wnt-PCP) pathway. In STAT3-depleted embryos, although all the known molecules in the Wnt-PCP pathway were expressed normally, the RhoA activity in lateral mesendodermal cells was down-regulated, resulting in severe cell polarization defects in convergence and extension movements identical to Strabismus-depleted embryos. Cell-autonomous activation of Wnt-PCP signaling by ΔN-dishevelled rescued the defect in cell elongation, but not the orientation of lateral mesendodermal cells in STAT3-depleted embryos. The defect in the orientation could be rescued by transplantation of shield cells having noncell-autonomous activity of STAT3 signaling. These results suggest that the cells undergoing convergence and extension movement may sense the gradient of signaling molecules, which are expressed in gastrula organizer by STAT3 and noncell-autonomously activate PCP signaling in neighboring cells during zebrafish gastrulation.